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General Remarks

In wheat and barley, tandem kinases (TKs) have emerged as a significant class of resistance determinants. Chen et al. previously cloned the TK Sr62TK and here they show it relies on a genetically linked NLR immune protein, Sr62NLR, to function. They propose a model in which the AvrSr62 pathogen effector binds the N-terminal kinase of Sr62TK (Kinase 1), disrupting its complex with the C-terminal kinase (Kinase 2), thus allowing it to activate Sr62NLR. A key finding is that Kinase 2 alone, without the full-length Sr62TK context, can activate Sr62NLR in the absence of the AvrSr62 effector. This result provides a unique perspective on the complex mechanisms underpinning this particular cereal immune system.

The paper is well-executed, with high-quality experiments and clear presentation. However, there are a few technical comments and missing controls. Nonetheless, it's a compelling narrative with significant findings.

Regarding the impact, it's noteworthy that the paper's findings confirm what many would have expected when TKs were first reported: Sr62TK is a direct decoy receptor for the pathogen effector, guarded by an NLR protein. This isn't entirely surprising, given that one of the earliest plant disease resistance genes, Pto, is also a kinase (albeit not a tandem kinase). Furthermore, kinases are generally co-receptors or integrated into NLR proteins, playing crucial roles in cell surface immunity through membrane receptor kinases and their downstream kinase partners. In another system, ZAR1 guards a decoy kinase, indicating that coevolution with bacterial pathogens occurs through the decoy kinases, not the NLR.

The manuscript could improve in terms of framing the mechanistic advancements in the context of NLR studies. The introduction, especially around page 2, lines 20-30, could better establish the conceptual framework for this research instead of listing the various kinases in barley and wheat, many of which aren't even TKs. A broader perspective on how this work aligns with established knowledge in plant immunity through decoy kinases (such as Pto, ZAR1-associated kinases, and other NLR-integrated kinases) would provide more context for readers than the laundry list of cereal immune kinases.

List of comments:

• Page 2, Line 10: The statement that many NLRs are autonomous is true, but somewhat misleading given that the majority require additional proteins (NLR helpers, guardees, or decoys) to function effectively.

• Figure 1 and AvrSr62: While the cloning of the AVR effector is critical to this work, it's not particularly groundbreaking in 2024, given the large number (hundreds) of cloned plant pathogen AVRs, including from rust pathogens. This limits the conceptual novelty of the study and perhaps this doesn’t deserve a main Figure (they could then expand on the mechanistic work).

• Complementation Experiments: The experiments where the researchers co-expressed three components are well-executed and provide convincing results.

• Fielder Sr62TK's Recognition of AvrSr62: It's interesting that Fielder's Sr62TK doesn't recognize AvrSr62, but the fact that Sr62TK works in Fielder suggests that the orthologue of Sr62NLR in Fielder is functionally conserved. They mention that the Fielder orthologue has 94.6% amino acid identity to its Ae. sharonensis counterpart. This claim should be supported with a supplementary figure.

• Figure 2d: An AlphaFold model of the NLR does not add anything to the discussion, the schematic diagram of figure 2c is sufficient for this figure and message.

• Given this observation, a phylogenetic analysis of Sr62NLR across grasses, especially in wheat, could add significant value, particularly since the ultimate goal and relevance of this work is to breed for Pgt resistance.

• Figure 3A: The split luciferase complementation assay requires critical controls where the proteins are expressed alone or in double combinations. These controls should include additional AvrSr62 as negative controls and western blot checks to confirm protein stability. This is important because certain combinations might affect protein stability, impacting the assay's results.

• Additionally, given that the combination of the three proteins leads to cell death, the assay and accuracy of the readout could very well be impacted.

• Figure S11: It's stated that co-expression using recognized AvrSr62 proteins leads to enhanced co-immunoprecipitation of Sr62NLR by Sr62TK. However, in Figure S11, the third and fourth bands have similar intensity for co-immunoprecipitated HA-Sr62NLR, even though only one of the two AvrSr62 effectors binds the TK. The slight increase in band intensity in lanes five and six requires replication and quantification to confirm its validity.

• Figure 3D: The self-association of full-length Sr62TK lacks an essential negative control for Sr62TK-FLAG. Including another FLAG-tagged protein as a control would add rigor to this experiment.

• Ponceau stains for the inputs of Co-IPs in figures 3D/F are required for a loading control.

• Figure S16: The claim that TK self-association was unaffected by the AvrSr62 proteins seems contradictory to their model, where the effector disrupts Kinase 2 in the full-length Sr62TK. This inconsistency needs clarification.

• Page 5, Line 1-2: Context is missing for the statement, “This effect was dependent on an intact alpha-1 helix in Sr62NLR (Figure S18B).” Providing more background or references would clarify this assertion.

• AlphaFold Predictions: The section on AlphaFold prediction can be shortened, especially in the absence of experimental validation for the predicted structure models. Including this as a main figure seems excessive without supporting data. Additionally, this section contains several assumptions that remain speculative. For example, it’s still unclear whether Sr62TK forms intramolecular complexes in addition to dimeric/multimeric complex that is then disrupted by AvrSr62. Additionally, although the Co-IPs performed indicate a homo-oligomeric complex is forming between the Sr62TK. Throughout the manuscript, the authors refer to Sr62TK as forming a dimer without any additional backing for this assumption. More supportive data is required to justify the assumptions.

• Figure S21: Clearer labelling of where the domains are would make the PAE plots for easier to interpret.

Competing interests

The authors declare that they have no competing interests.